Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Dec 8:4:7358.
doi: 10.1038/srep07358.

Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods

Yuhua Wu  1 , Yulei Wang  2 , Jun Li  1 , Wei Li  1 , Li Zhang  3 , Yunjing Li  1 , Xiaofei Li  1 , Jun Li  1 , Li Zhu  1 , Gang Wu  1
Affiliations

Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods

Yuhua Wu et al. Sci Rep. .

Abstract

The Cauliflower mosaic virus (CaMV) 35S promoter (P35S) is a commonly used target for detection of genetically modified organisms (GMOs). There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON88913 cotton, and the other two methods could only be applied to certain GMOs. The rest three reported methods were not suitable for measurement of P35S in some testing events, because SNPs in binding sites of the primer/probe would result in abnormal amplification plots and poor linear regression parameters. In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events, and developed new qualitative and quantitative detection systems targeting this conserved region. The qualitative PCR could detect the P35S promoter in 23 unique GMO events with high specificity and sensitivity. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event. This study provides a general P35S screening method, with greater coverage than existing methods.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Sequence alignment of the P35S conserved region from CaMV, transgenic events and binary vectors harboring SNPs, together with primer/probe sets of the M2, M7, M12 methods and primer/probe set (M25) designed in this study.
Figure 2
Figure 2. PCR amplification of P35S targets in GM crops using existing methods.
(a) Amplification of the P35S fragment in MON 88913 event using the 24 existing P35S-based methods, Lanes 1–24 correspond to the M1-M24 methods. (b–c) Amplification of the P35S fragments in 23 transgenic events by methods M10 and M18, respectively. Lanes 1–24 correspond to the following samples: GM soybean (1) GTS40-3-2, (2) A5547-127, (3) A2704-12; GM maize (4) Bt11, (5) TC1507, (6) T25, (7) Bt176, (8) NK603, (9) MON89034, (10) M88017, (11) MON810, (12) MON863, (13) 59122; GM cotton (14) MON88913, (15) MON1445, (16) MON531, (17) LLcotton25, (18) MON15985; GM rapeseed (19) T45, (20) Topas19/2, (21) OXY235; GM rice (22) Kefeng 6, (23) KMD; (24) non-GM crop mixture (soybean, maize, cotton, rapeseed and rice).
Figure 3
Figure 3. TaqMan assays using M2, M7, and M12 methods with serial DNA dilutions from events MON810, TC1507, Bt11, and the vector pMCG161.
Standard curves were constructed based on the amplification plot. MON810 maize, which does not contain a SNP, was used as the control. Methods M2, M7 and M12 were assessed with events TC1507, Bt11, and vector pMCG161.
Figure 4
Figure 4. Testing of the amplification stability and sensitivity of the qualitative PCR detection.
(a) Amplification of P35S in different GM crops. Key: Lane M, DL 1000 DNA Marker, Lanes 1–24 correspond to the following samples: GM soybean (1) GTS40-3-2, (2) A5547-127, (3) A2704-12; GM maize (4) Bt11, (5) TC1507, (6) T25, (7) Bt176, (8) NK603, (9) MON89034, (10) M88017, (11) MON810, (12) MON863, (13) 59122; GM cotton (14) MON88913, (15) MON1445, (16) MON531, (17) LLcotton25, (18) MON15985; GM rapeseed (19) T45, (20) Topas19/2, (21) OXY235; GM rice (22) Kefeng 6, (23) KMD; (24) non-GM crop mixture (soybean, maize, cotton, rapeseed and rice). (b) Sensitivity of the qualitative PCR method. Serially diluted DNA extracts of maize TC1507, soybean GTS 40-3-2, cotton MON 1445, rice Kefeng 6, and rapeseed OXY235 were used as templates. Lanes 1–4 correspond to 100, 50, 20, and 10 haploid genome copies, respectively; each template was run with two parallel PCR reactions.
Figure 5
Figure 5. Amplification plot and standard curves for real-time quantitative PCR assays of P35S using serially diluted genomic DNA from five transgenic events as calibrators.
(a–e) correspond to the amplification plots and standard curves of events GTS-40-3-2, MON1445, OXY235, KMD1, and TC1507, respectively.
See this image and copyright information in PMC

References

    1. Bridgers M. Genetically modified organisms and the precautionary principle: how the GMO dispute before the world trade organization could decide the fate of international GMO regulation. Temp. Envtl. L. Tech. J. 22, 171 (2004).
    1. Marmiroli N. et al. Methods for detection of GMOs in food and feed. Anal Bioanal Chem. 392, 369–384 (2008). - PubMed
    1. Holst-Jensen A., Rønning S. B., Lovseth A. & Berdal K. G. PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal. Bioanal. Chem. 375, 985–993 (2003). - PubMed
    1. Holst-Jensen A. Testing for genetically modified organisms (GMOs): Past, present and future perspectives. Biotechnol. Adv. 27, 1071–1082 (2009). - PubMed
    1. James C. Global status of commercialized biotech/GM crops: 2013. ISAAA Briefs No. 46 http://www.isaaa.org/purchasepublications/itemdescription.asp?ItemType=B... (2013) Date of access: 5 Apr. 2014.

Publication types

LinkOut - more resources